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Scientists have been manipulating genes in test tubes for years. Doing the same in living organisms, however, is a different story: It requires a way to precisely target that gene. A mismatch could mean the difference between effectively treating a disease and triggering the development of cancer. Two HHMI investigators recently teamed up to test the accuracy of a molecule called Cas9 that can be instructed to bind to and modify specific genes.

Cas9 is a naturally occurring bacterial protein with a bright future in genome engineering and gene therapy. It can be programmed with a short piece of RNA to cut specific DNA sequences. The RNA “guide” is usually about 20 base pairs long and is complementary to the target DNA. David Liu of Harvard University, Jennifer Doudna of the University of California, Berkeley, and their students evaluated Cas9’s ability, when paired with guide RNAs of different lengths, to cut one trillion different DNA sequences.

They found that while the entire guide RNA sequence can program Cas9 specificity, some genome sequences that do not exactly match the guide RNA can nevertheless be cleaved, according to the report in the September 2013 issue of Nature Biotechnology. The tolerance for mismatches depends on what site in the genome was targeted and where the mismatches occurred in the guide RNA sequence. The team also found a trade-off between activity and specificity. Shorter, less active guide RNAs are more specific than longer, more active ones. Doudna and Liu are continuing their collaboration, which is now aimed at improving Cas9 specificity. They believe that, with future improvements, the molecule should be able to home in on any single site in the human genome, with potential therapeutic implications.